(a) Field of the Invention
The present invention relates to a saccharide-based detection sensor and its use, which detection sensor is to detect the existence of Vibrio cholerae in a sample by detecting a cholerae toxin as a Vibrio cholerae detection marker. More particularly, the present invention relates to a carbohydrate chip for detection of Vibrio cholerae, a method for detecting Vibrio cholerae using the same, and a method for preparing the same, where GM1 pentasaccharide, GM2 pentasaccharide, asialo GM1 tetrasaccharide, GM3 trisaccharide, galactose-β 1,3-N-acetylgalactosamine, lactose, and sialic acid are immobilized on the surface of a solid substrate.
(b) Description of the Related Art
The present invention is directed to a technique for detecting pathogen Vibrio cholerae that causes cholera, by means of a micro array (chip) which can be used to analyze and detect the interactions of different organic molecules simultaneously from a small quantity of sample.
A representative pathogenic bacterium, Vibrio cholera is a Gram-negative bacterium with a single, polar flagellum and belongs to the gamma subdivision of the Proteobacteria. Vibrio cholerae is a bacterium causing cholera which still remains a pervasive threat in the Earth. Left untreated, cholera can be fatal with high death rate, averaging 40 to 50%. Hence, the detection and diagnosis of the pathogenic bacterium Vibrio cholerae is a field very critical to the welfare of the human beings, and many efforts have been made to establish it.
Vibrio cholerae releases a substance called “cholera toxin” which causes cholera disease (De Haan, L, Hirst, T R, 2004. Mol. Membr. Biol. 21: 77-92). Hence, the cholera toxin is used as a detection marker for Vibrio cholerae in the Vibrio Cholerae detection system.
More specifically, cholera toxin is a protein enterotoxin secreted by pathogen Vibrio cholerae that is the major causative agent of cholera and used as a detection marker of Vibrio cholerae. Cholera toxin consists of one A subunit and five B subunits. The toxin B subunits bind specifically to GM1 gangliosides on the surface of endothelial cells to help uptake of the toxin A subunit into cells. The toxin A subunit possesses enzymatic (ADP ribosyltransferase) activity involved in accumulation of cAMP in the cells (Fukuta, S, Magnani, J L, Twiddy, E M, Holmes, R K, Ginburg, V, 1998. Infect. Immun. 56: 1748-1753).
There have been developed many detection methods for Vibrio cholerae, mostly based on antibodies or DNA. Antibody-based techniques for detecting Vibrio cholerae include enzyme-linked immunoassorbent assay (ELISA), surface plasma resonance (SPR), quartz crystal microbalance (QCM), microcantilever-based immunosensor, electrochemical immunosensor, and so forth. DNA-based techniques for detecting Vibrio cholerae include polymerase chain reaction (PCR), real time PCR (RT-PCR), multiplex PCR, DNA probe hybridization, and so forth. These techniques are characterized by high sensitivity for detection of Vibrio cholerae but problematic in stability as a sensor for detecting Vibrio cholerae due to a decrease in activity caused by denaturation of antibodies and degradation of DNA by DNase. The antibody-based sensors for detection of Vibrio cholerae has a disadvantage that it requires advanced techniques for immobilizing antibodies with orientation.
Accordingly, there have recently been developed saccharide-based sensors for detection of Vibrio cholerae using the fact that cholera toxin secreted by Vibrio cholerae specifically binds to GM1 gangliosides on the surface of endothelial cells. The saccharide-based sensors for detection of Vibrio cholerae are known to have some advantages over the existing antibody- or DNA-based sensors for detection of Vibrio cholerae (Chen, H, Zheng, Y, Jiang, J-H, Wu, H-L, Shen, G-L, Yu, R-Q, 2008. Biosens. Bioelectron. 24: 684-689). Firstly, GM1 pentasaccharide has the higher structural stability than antibodies, so the sensors for detection of Vibrio cholerae using GM1 pentasaccharide are superior in stability to the antibody-based sensors for detection of Vibrio cholerae. Secondly, GM1 gangliosides have strong affinity to cholera toxin. Lastly, the combining process of GM1 gangliosides and cholera toxin occurs very rapidly.
Recent advances in saccharide-based sensors for detecting Vibrio cholerae have been reported. Some of the sensors are based on the GM1 gangliosides and their analogues immobilized on the surface coated with a lipid film such as a membrane and liposome functionally surface-treated with the saccharides, using a variety of analytical tools, such as surface plasmon resonance (Phillips, K S, Han, J H, Martinez, M, Wang, Z Z, Carter, D, Cheng, Q, 2006. Anal. Chem. 78: 596-603), fluorescent quenching (Song, X, Swanson, B I, 1999. Direct, Anal. Chem. 71: 2097-2107), resonant energy transfer (Ma, G Y, Cheng, Q, 2006. Langmuir 22: 6743-6745), and so forth. These methods are, however, disadvantageously inferior in detection sensitivity.
Some methods are based on the liposomes surface-treated with GM1-gangliosides and their analogues, which liposomes are processed to contain fluorescent reagents (Ho, J A, Wu, L C, Huang, M R, Lin, Y J, Baeumner, A J, Durst, R A, 2007. Anal. Chem. 79: 246-250), colorimetric markers (Ahn-Yoon, S, Decory, T R, Baeumner, A J, Durst, R A, 2003. Anal. Chem. 75: 2256-2261), enzyme (Alfonta, L, Singh, A K, Willner, I, 2001. Anal. Chem. 73: 91-102), electroactive species (Viswanathan, S, Wu, L C, Huang, M R, Ho, J A, 2006. Anal. Chem. 78: 1115-1121), etc. These sensors have high sensitivity in relation to the preceding methods, but the liposome-based sensors for detection of Vibrio cholerae take long time in their fabrication due to complicity of steps and feature some problems in regard to low repeatability and poor stability.
Some studies have been made on the analysis of interactions between GM1 gangliosides and cholera toxin using a microarray based on GM1 gangliosides (Fang, Y, Frutos, A G, Lahiri, J, 2003. Langmuir 19: 1500-1505; Ngundi, M M, Taitt, C R, McMurry, S A, Kahne, D, Ligler, F S, 2006. Biosens. Bioelectron. 21: 1195-1201; and Zhang, J, Zhou, X, 2011. Biosens. Bioelectron. 28: 355-361). However, the analysis in the studies uses GM1 gangliosides and their analogues rather than their carbohydrate moiety as a capture probe, and the liposome used to immobilize the gangliosides has some problems, such as change of structure, non-uniform particle size, aggregation and fusion, and chemical instability, resulting in difficulty and poor repeatability of immobilization. Poor repeatability in the immobilization process may be a serious disadvantage to the sensor for detection of Vibrio cholerae. 
To solve the problems with liposome used to immobilize gangliosides, the present invention has demonstrated that the carbohydrate chips in which the gangliosides-constituting carbohydrates are immobilized on a solid substrate through chemical modification and covalent bonding is applicable to the analysis of interactions between cholera toxin and carbohydrates constituting the gangliosides and the detection of Vibrio cholerae using cholera toxin. In addition, the present invention also uses GM2 tetrasaccharide, asialo GM1 tetrasaccharide, GM3 trisaccharide, galactose-β 1,3-N-acetylgalactosamine, lactose, and sialic acid, in combination with GM1 ganglioside mainly used in the conventional saccharide-based sensors for detection of Vibrio cholerae, thereby detecting a low concentration of cholera toxin which is a detection marker for Vibrio cholerae and even the cholera toxin actually produced by Vibrio cholerae. 